The proinflammatory cytokine interleukin-1 (IL-1) functions in the generation of systemic and local responses to infection, injury and immunological challenges. The importance of IL-1 in inflammation has been demonstrated by the ability of the highly specific IL-1 receptor antagonist protein (IL-1Ra, or IRAP) to relieve inflammatory conditions (for review, see, e.g., Dinarello, Cytokine Growth Factor Rev. 8:253-265 (1997)). IL-1 is produced primarily by activated macrophages and monocytes, and is involved in lymphocyte activation, fever, leukocyte trafficking, the acute phase response, cartilage remodeling and other processes. IL-1 exerts its effects by binding to a receptor, IL-1RI, located on the plasma membrane of responsive cells. Among the results of IL-1 binding to the IL-1 RI receptor is the activation of the NF-xcexaB transcription factor, ultimately leading to the expression of numerous genes involved in inflammation, such as cytokines, growth factors, immunoreceptors, and cell adhesion molecules (for review, see, e.g., Lee, et al., J. Clin. Pharmacol. 38(11):981-93 (1998)).
Several proteins have been discovered to mediate signal transduction following IL-1RI activation, ultimately leading to the activation of NF-KB. For example, the IL-1R accessory protein, IL-1RAcP, has been shown to associate with the IL-1RI receptor following binding to IL-1, thereby initiating the signal transduction cascade (Greenfeder, et al., J. Biol. Chem. 270(23):13757-65 (1995)). In addition, three IL-1 receptor-associated kinases (IRAKs) have been identified, IRAK (xe2x80x9cIRAK-1;xe2x80x9d Cao, et al., Science 271:1128-1131 (1996)), IRAK-2 (Muzio, et al., Science 278:1612-1615 (1997)), and the monomyeloic cell-specific IRAK-M (Wesche, et al., J. Biol. Chem. 274:19403-10 (1999)). IRAK has been shown to be phosphorylated and to associate with IL-1RI in an IL-1 dependent manner. In addition, the MyD88 protein has been shown to mediate the association of IRAK proteins to the activated IL-1 receptor (Wesche, et al., 7:837-47 (1997)). Also, TRAF6 transduces the IRAK signal to downstream effector molecules (Cao, et al., Nature 383:443-6 (1996)). The IRAK proteins, as well as MyD88, have been shown to play a role in transducing signals other than those originating from IL-1R receptors, including signals triggered by activation of IL-18 receptors (Kanakaraj, et al. J. Exp. Med. 189(7):1129-38 (1999)) and LPS receptors (Yang, et al., J. Immunol. 163:639-643 (1999); Wesche, et al., J. Biol. Chem. 274:19403-10 (1999)). Overexpression of IRAK-2 and IRAK-M has been shown to be capable of reconstituting the response to IL-1 and LPS in an IRAK deficient cell line.
The IL-1 signal transduction cascade is analogous to a signaling cascade in Drosophila melanogaster that is involved in the establishment of dorsal ventral polarity during the early development of Drosophila embryos. Specifically, in Drosophila, the extracellular ligand Spaetzle binds to a receptor called Toll, which shares homology to IL-1R. In addition, a serine/threonine kinase acting downstream of Toll activation, Pelle is homologous to IRAK kinases (Cao, et al., Science 271:1128-1131 (1996); Muzio, et al., Science 278:1612-1615 (1997); Wesche, et al., J. Biol. Chem. 274:19403-19410 (1999)). Finally, activation of the Toll receptor results in the activation of the transcription factor Dorsal, which is homologous to NF-xcexaB. Dorsal is inhibited in Drosophila cells by Cactus, which is itself homologous to the NF-xcexaB inhibitor IxcexaB.
The present invention is based on the identification of a novel member of the IRAK family, IRAK-4. Nucleic acid and protein sequences for IRAK-4 are provided, as are methods of making IRAK-4 nucleic acids and proteins. Also provided are methods of using IRAK-4 polynucleotides and polypeptides, including methods of using the herein-disclosed sequences to isolate compounds useful in the treatment or prevention of any of a number of inflammatory diseases and conditions.
The present invention provides novel nucleic acids and polypeptides for mammalian IRAK-4, a new member of the IRAK gene family. IRAK kinases associate with activated IL-1, IL-18 and other receptors and act to transduce signals originating from the activated receptors, ultimately leading to a variety of downstream effects such as NF-xcexaB activation.
In one aspect, an isolated nucleic acid is provided encoding an IRAK-4 polypeptide, the polypeptide comprising at least about 98% amino acid sequence identity to SEQ ID NO:1 or to a subsequence thereof, wherein the amino acid sequence of the polypeptide comprises an alanine residue at an amino acid position corresponding to amino acid position 81 of SEQ ID NO:1, and wherein said nucleic acid comprises at least about 400 nucleotides.
In one embodiment, the polypeptide further comprises an amino acid selected from the group consisting of: (i) a valine residue at an amino acid position corresponding to amino acid position 432 of SEQ ID NO:1; (ii) a leucine residue at an amino acid position corresponding to amino acid position 437 of SEQ ID NO:1; (iii) an arginine residue at an amino acid position corresponding to amino acid position 444 of SEQ ID NO:1; and (iv) a glutamine residue at an amino acid position corresponding to amino acid position 451 of SEQ ID NO:1. In another embodiment, the polypeptide comprises each of the amino acids listed as (i) to (iv). In another embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises at least about 100 amino acids. In another embodiment, the polypeptide comprises at least about 450 amino acids.
In another embodiment, the nucleic acid comprises a cytosine at a nucleotide position corresponding to nucleotide position 242 of SEQ ID NO:2. In another embodiment, the nucleic acid further comprises a nucleotide selected from the group consisting of: (i) a thymine at a nucleotide position corresponding to nucleotide position 1295 of SEQ ID NO:2; (ii) a thymine at a nucleotide position corresponding to nucleotide position 1302 of SEQ ID NO:2; (iii) a thymine at a nucleotide position corresponding to nucleotide position 1310 of SEQ ID NO:2; (iv) an adenine at a nucleotide position corresponding to nucleotide position 1332 of SEQ ID NO:2; and (v) an adenine at a nucleotide position corresponding to nucleotide position 1353 of SEQ ID NO:2. In another embodiment, the nucleic acid comprises each of the nucleotides listed as (i) to (v). In another embodiment, the nucleic acid comprises a nucleotide sequence of SEQ ID NO:2. In another embodiment, the nucleic acid comprises at least about 350 nucleotides. In another embodiment, the polypeptide specifically binds to antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:1.
In another aspect, the present invention provides an isolated IRAK-4 polypeptide, the polypeptide having at least about 98% amino acid sequence identity to SEQ ID NO:1 or to a subsequence thereof, wherein the amino acid sequence of the polypeptide comprises an alanine residue at an amino acid position corresponding to amino acid position 81 of SEQ ID NO:1, and wherein the polypeptide comprises at least about 100 amino acids.
In one embodiment, the polypeptide further comprises an amino acid selected from the group consisting of: (i) a valine residue at an amino acid position corresponding to amino acid position 432 of SEQ ID NO:1; (ii) a leucine residue at an amino acid position corresponding to amino acid position 437 of SEQ ID NO:1; (iii) an arginine residue at an amino acid position corresponding to amino acid position 444 of SEQ ID NO:1; and (iv) a glutamine residue at an amino acid position corresponding to amino acid position 451 of SEQ ID NO:1. In another embodiment, the polypeptide comprises all of the amino acids listed as (i) to (iv). In another embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide is encoded by a nucleic acid comprising a nucleotide sequence of SEQ ID NO:2. In another embodiment, the polypeptide specifically binds to antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises at least about 450 amino acids.
In another aspect, the present invention provides an isolated nucleic acid encoding an IRAK-4 polypeptide, the polypeptide comprising at least about 70% amino acid sequence identity to SEQ ID NO:3 or to a subsequence thereof.
In one embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO:3. In another embodiment, the nucleic acid comprises at least about 70% nucleotide sequence identity to SEQ ID NO:4 or to a subsequence thereof. In another embodiment, the nucleic acid comprises a nucleotide sequence of SEQ ID NO:4. In another embodiment, the nucleic acid hybridizes under stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:4.
In certain embodiments, the above nucleic acids are operably linked to a promoter. In other aspects, the present invention provides expression cassettes comprising the nucleic acids, wherein the nucleic acids are operably linked to a promoter. In other aspects, the present invention provides isolated cells comprising an expression cassette.
In another aspect, the present invention provides a method of making an IRAK-4 polypeptide, the method comprising: (i) introducing a nucleic acid into a host cell or cellular extract, the nucleic acid encoding a polypeptide comprising either: (a) at least about 98% amino acid sequence identity to SEQ ID NO:1 or to a subsequence thereof, wherein the polypeptide comprises an alanine residue at an amino acid position corresponding to amino acid position 81 of SEQ ID NO:1, and wherein the nucleic acid comprises at least about 400 nucleotides; or (b) at least about 70% amino acid sequence identity to SEQ ID NO:3 or to a subsequence thereof; (ii) incubating said host cell or cellular extract under conditions such that the IRAK-4 polypeptide is expressed in the host cell or cellular extract; and (ii) recovering the IRAK-4 polypeptide from the host cell or cellular extract.
In another aspect, the present invention provides a method of identifying a compound useful in the treatment of inflammatory diseases, the method comprising the steps of: (i) contacting an IRAK-4 polypeptide with the compound, wherein the IRAK-4 polypeptide comprises at least about 70% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3; and (ii) determining the functional effect of the compound on the IRAK-4 polypeptide.
In one embodiment, the IRAK-4 polypeptide comprises an amino acid sequence shown as SEQ ID NO:1 or SEQ ID NO:3. In another embodiment, the compound inhibits IRAK-4 kinase activity. In another embodiment, the IRAK-4 is present inside of a eukaryotic cell.
In another aspect, the present invention provides a method of treating an inflammatory disease in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound identified using the method comprising the steps of: (i) contacting an IRAK-4 polypeptide with the compound, wherein the IRAK-4 polypeptide comprises at least about 70% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3; and (ii) determining the functional effect of the compound on the IRAK-4 polypeptide.
In one embodiment, the inflammatory disease is selected from the group consisting of pulmonary diseases and diseases of the airway, transplant rejection, autoimmune diseases, cancer, cardiovascular diseases, diseases of the central nervous system, CD14 mediated sepsis, non-CD14 mediated sepsis, osteoarthritis, osteoporosis, psoriasis, diseases of the skin, inflammatory bowel disease, Behcet""s syndrome, ankylosing spondylitis, sarcoidosis, gout, and ophthalmic diseases and conditions.
In one embodiment, the pulmonary disease and disease of the airway is selected from the group consisting of Adult Respiratory Disease Syndrome (ARDS), Chronic Obstructive Pulmonary Disease (OPD), pulmonary fibrosis, interstitial lung disease, asthma, chronic cough, and allergic rhinitis. In another embodiment, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and diabetes (e.g., type 1 diabetes mellitus). In another embodiment, the cancer is selected from the group consisting of solid tumors, skin cancer, and lymphoma. In another embodiment, the cardiovascular disease is selected from the group consisting of stroke and atherosclerosis. In another embodiment, the disease of the central nervous system is a neurodegenerative disease. In another embodiment, the disease of the skin is selected from the group consisting of rash, contact dermatitis, and atopic dermatitis. In another embodiment, the inflammatory bowel disease is selected from the group consisting of Crohn""s disease and ulcerative colitis.
In another aspect, the present invention provides a method of inhibiting the transduction of a signal resulting from the activation of an IL-1R/Toll receptor in a cell, the method comprising introducing into the cell an inhibitor of the activity or expression of IRAK-4.
In one embodiment, the IL-1R/Toll receptor is activated by IL-1. In another embodiment, the inhibitor comprises a dominant negative form of IRAK-4. In another embodiment, the dominant negative form of IRAK-4 comprises a mutation in a lysine residue in the ATP binding pocket. In another embodiment, the mutation comprises a substitution of alanine residues for lysine residues within the IRAK-4 at amino acid positions corresponding to positions 213 and 214 of SEQ ID NO:1. In another embodiment, the dominant negative form of IRAK-4 is a truncated form of IRAK-4. In another embodiment, the truncated form of IRAK-4 consists essentially of amino acids 1 to 191 of SEQ ID NO:1. In another embodiment, the inhibitor comprises a compound identified using the method comprising the steps of: (i) contacting an IRAK-4 polypeptide with the compound, wherein the IRAK-4 polypeptide comprises at least about 70% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3; and (ii) determining the functional effect of the compound on the IRAK-4 polypeptide. In one embodiment, the inhibitor inhibits activation of at least one transcription factor. In another embodiment, the transcription factor activates NFxcexaB in the cell.
In another aspect, the present invention provides a transgenic nonhuman animal that comprises a mutation in an endogenous IRAK-4 gene. In one embodiment, the mutation inactivates the endogenous IRAK-4 gene. In another embodiment, the mutation deletes all or part of the IRAK-4 gene. In another embodiment, the animal is a mouse.
In another aspect, the present invention provides an isolated mammalian cell comprising a mutation in an endogenous IRAK-4 gene. In one embodiment, the mutation inactivates the IRAK-4 gene. In another embodiment, the mutation deletes all or part of the IRAK-4 gene.